Structural biology and search for inhibitors of the non-structural protein NS5 RNA-dependent RNA polymerase domain of the Zika virus

Author(s)

Ketllyn Irene Zagato de Oliveira

• Document type: Doctoral Thesis
• Press: São Carlos.
• Institution: Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT)
• Defense date: 2021-04-07

Examining board members

Glaucius Oliva; Marcio Vinícius Bertacine Dias; Andrea Balan Fernandes; Ana Carolina Gomes Jardim; Dulce Helena Ferreira de Souza

• Advisor: Glaucius Oliva

Abstract

The Zika virus (ZIKV) is a member of the Flaviviridae family that was first reported in Brazil in April 2015. The major impact of ZIKV was due to its association with the increase in the number of microcephaly cases reported in newborns of pregnant women infected with the virus during the epidemic that occurred in Brazil between 2015 and 2016. Since then, more than 240.000 cases of Zika fever have been reported in the country, with 10.768 cases registered in 2019 and 6.705 probable cases in 2020. Given these facts, many studies are underway to search for candidates for effective drugs against ZIKV. Among the ZIKV non-structural proteins, NS5 is of great interest as a pharmacological target, due to the existence of an inhibitor approved for clinical use against the Hepatitis C virus NS5 RNA-dependent RNA polymerase domain (RdRp). RdRp is responsible for the de novo synthesis of viral RNA, that is, without the need for a primer to start the synthesis. It promotes the elongation of viral RNA, through polymerization using NTPs, thus guaranteeing viral replication. Inhibition of any of these functions would prevent viral replication. In this context, the objective of this project was to obtain the NS5 RdRp domain from the ZIKV in order to determine its three-dimensional structure by X-ray crystallography, followed by the discovery of inhibitors. For this purpose, we designed two constructions for the NS5 RdRp protein: the RdRp_truncated and the RdRp_integral. With the RdRp_truncated, it was possible to obtain crystals that produced X-ray diffraction, which enabled the elucidation of the crystallographic structure of the NS5 RdRp domain at 1.9 Å resolution. This structure was used to perform a computational screening of inhibitors as well as biophysical assays to search for possible ligands. With RdRp_integral, it was possible to standardize an assay based on the fluorescent nucleic acid marker SYBR Green I, which monitors the activity of the protein in real time, and which was used in the extended search for inhibitors. It was possible to test the inhibition of more than 500 compounds, including the drug-like compound library Pandemic Response Box (PRB, Medicines for Malaria Venture - MMV), as well as synthetic compounds and natural products that were previously identified by biophysical methods and virtual screening. In all, 17 potential NS5 RdRp inhibitors were identified, with 9 of them showing inhibition in the micromolar range (IC50 ≤10 μM) and 5 of them showing inhibition in the nanomolar range (IC50 ≤1 μM). The results obtained in this work demonstrate that despite the absence of crystallographic structures complexed with ligands, it was possible to find molecules with high inhibition, which can have their action optimized by computational approaches and subsequently tested against the ZIKV NS5 RdRp. Among them, the natural products that have shown good RdRp inhibitory activity seem to be very promising for further studies, particularly those derived from the flavonol ring. (AU)

• FAPESP's process: 18/19574-0 - Structural biology and drug development against the protein NS5 Zika Virus
• Grantee: Ketllyn Irene Zagato de Oliveira
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)